This proposal is to perform structure/function studies, by solution phase nuclear magnetic resonance spectroscopy, of the 29 kDa C-terminal domain of bacteriophage T4 primase. This domain comprises both the catalytic RNA polymerase activity and the helicase interacting domain of the primase. By incorporating both activities, this construct should reveal structure/function relationships while remaining accessible for study. A robust and straightforward expression protocol yields NMR quality samples from 1 L M9 preparations (minimal media), and 1H{15N} trosy-hsqc experiments conducted at 600 MHz indicate a folded protein that appears to be a strong candidate for structure determination and investigation. Doubly enriched samples (13C,15N) have been prepared and used to obtain several multi-dimensional NMR experiments employing trosy and non-uniform sampling. The only structural information on T4 primase to date has been obtained from electron microscopy on functional hexamers formed by T4 primase and no molecular level structural information is known at this time. Initial backbone assignments will reveal secondary structure and create opportunities for interaction mapping by chemical shift perturbations and by the measurement of backbone dynamics. The binding of the C-terminal domain to ribonucleotides and oligoribonucleotides will be investigated both by chemical shift perturbations of the backbone atoms, and possibly by NOE analysis. A full structure will be pursued by modern techniques in NMR spectroscopy, which would reveal the catalytic active site for the first time, and would stimulate a variety of new inquiries into T4 function. Chemical shift assignments of the C-terminal domain will be used to obtain backbone assignments of the full length T4 primase, which also expresses well and which includes only an additional 10 kDa zinc-binding domain. Full-length T4 primase (40 kDa) will be investigated in the later stages of the work, focusing initially on obtaining chemical shift assignments and detecting chemical shift perturbations to further probe interactions (i) between T4 primase and (oligo)ribonucleotides to gain insight into conformational changes and into regulation of RNA primer length and (ii) between T4 primase and target ssDNA in the hopes of revealing mechanisms for target site recognition. PUBLIC HEALTH RELEVANCE: This proposal is to study the structure and function of a specific primase protein, which is involved in DNA replication for the prokaryotic bacteriophage T4. The process by which cells carry out DNA replication is very complex in eukaryotic organisms and, only by comparison, simpler in prokaryotic systems such as bacteria and their phages. Understanding prokaryotic DNA replication in general will extend knowledge of the process of bacterial replication, which is targeted by some antibiotics, and may help to bridge the gap to understanding eukaryotic (e.g. human) replication. Any fundamental information on DNA replication in humans could provide basic insights on mechanisms of cancer. Bacteriophage T4 is a good model system for DNA replication and the proposed work will study the primase component of T4. No structural information on the molecular level is known for any part of the T4 primase. The proposed work will reveal new structural information on T4 primase and will lay out a pathway for structural studies of larger parts of the T4 replication system.